Establishment of an axis of cell polarity is fundamental for normal development of many types of cells and tissues. In many cases, establishment of cell polarity is intimately linked to progression through the cell cycle, and therefore may be relevant to cancer progression. Bud site selection in the yeast S. cerevisiae provides a powerful model system to study cell polarity at a molecular level. S. cerevisiae cells bud at defined spots on their cell surfaces, thought to be marked by proteins, such as Bud8. The marker is recognized by a signaling cascade which causes the cytoskeleton to orient toward that site. Ste2O is one of the kinases involved in polarity signaling to the cytoskeleton. It has recently been shown that Ste2O is specifically required for budding from the distal pole, and is the only protein known to be required specifically for distal budding in diploids other than Bud8 itself (Sheu et al., 2000). This raises the possibility that Ste20 activates Bud8, perhaps via phosphorylation. Consistent with this possibility, Bud8 runs on SDS-PAGE as a doublet (Taheri et al., 2000) To test this hypothesis, in vivo Bud8 phosphorylation will be characterized in detail, and its dependence upon Ste20 function will be tested. Furthermore, the functional consequences of Bud8 phosphorylation will be tested using specific alanine and aspartate mutants and assaying bud site selection, Bud8 localization, and other phenotypes relevant to Bud8. In addition to possibly phosphorylating Bud8, Ste20 may regulate Bud8 s localization and/or abundance. Therefore, Bud8 localization and abundance will be examined in a ste20delta background. Finally, nearly all of the protein kinases present in yeast will be screened for their ability to phosphorylate Bud8 in vitro using a protein kinase chip assay to identify potential direct regulators of Bud8.